The Tongue Squamous Carcinoma Cell Line Cal27 Primarily Employs Integrin α6β4-Containing Type II Hemidesmosomes for Adhesion Which Contribute to Anticancer Drug Sensitivity

Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVβ3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVβ3 expressing Cal27-derived cell clone 2B1, αVβ5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6β4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6β4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6β4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVβ3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6β4 not occurred. Taken together, our results identify a key role for α6β4-containing type II hemidesmosomes in regulating anticancer drug sensitivity

Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression

Electrophysiological analysis of rat renal sugar and amino acid transport. IV. Basic amino acids

Electrophysiological techniques were used to study the transport of the basic amino acids L-arginine, L-lysine and L-ornithine in rat kidney proximal tubule in vivo. Tubular cells were punctured with microelectrodes and the response of the cell membrane potential to sudden applications of the amino acids was measured. In the presence of physiological Na+ concentrations luminal perfusion with millimolar concentrations of basic amino acids depolarized the tubular cells in a concentration dependent fashion by up to 15 mV, while in the absence of Na+ no significant potential changes were observed. These observations indicate that the basic amino acids are taken up into the cell across the brushborder in coupling with Na+ ions in a similar way as neutral and acidic amino acids, and that simple conductive pathways for uncoupled flow of the basic amino acids do either not exist or are quantitatively negligible in the brushborder. From kinetic measurements and competition experiments it was concluded that all basic amino acids are transported by the same transport system, which however does not accept acidic or neutral amino acids (with the possible exception of L-cystine). Perfusion of the peritubular capillaries with millimolar concentrations of basic amino acids depolarized the cells only by approximately 1 mV, both in the presence and absence of Na+. This observation may indicate that a passive uncoupled transport pathway for basic amino acids is present in the peritubular cell membrane to allow exit from cell to interstitial space, if the intracellular concentration rises high enough to overcome the cell membrane potential

Electrophysiological analysis of rat renal sugar and amino acid transport. III. Neutral amino acids

We have used electrophysiological techniques in rat kidney proximal tubule to study the transport of the followingl-α-amino acids: alanine, phenylalanine, glutamine, methionine, cysteine, cystine, proline, and hydroxyproline as well as the transport of glycine and β-alanine. When applied in millimolar concentrations in the tubular lumen in the presence of Na+, all amino acids tested were found to depolarize the tubular cell membranes partially. This depolarization arises from current flow that is associated with the cotransport of Na+ and amino acids across the brushborder membrane (Frömter 1982 [9]). Peritubular application did not alter the membrane potentials in a conclusive way. The magnitude of the depolarization in response to luminal perfusion increased with increasing amino acid concentration and obeyed simple Michaelis-Menten kinetics, except for proline, hydroxyproline, and glycine, which exhibited double-site saturation kinetics. By analyzing the time course of the potential changes and distinguishing between initial and steady state depolarizations, it was possible to separate kinetic properties of the brushborder transport mechanisms from the lumped kinetic properties of the overall epithelium. Knowing the concentration dependence of the depolarizations, the competition of different amino acids for the same transport site was investigated: Two amino acids were applied in saturating concentrations in the tubular lumen, either singly or jointly, to determine whether the depolarizations were additive and whether the additivity exceeded the predictions from the kinetic experiments. From such studies the presence of three separate rheogenic transport systems is postulated for neutral amino acids: System I transports all neutrall-α-amino acids with the exception of cystine, system II transports proline, hydroxyproline and glycine, and system III transports β-alanine or under physiological conditions taurine. Studies with the oxidant diamide suggest that cystine may be mostly reduced to cysteine and transported as cysteine, however it is likely that an extra transport system exists which transports cystine possibly in electroneutral fashion together with dibasic amino acids

Electrophysiological analysis of rat renal sugar and amino acid transport. V. Acidic amino acids

We have used electrophysiological techniques to study various aspects of the transport of glutamate and aspartate in proximal tubules of the rat kidney in vivo. Single tubular cells were punctured with microelectrodes and the response of the cell membrane potential to sudden luminal or peritubular applications of these amino acids was measured. The experiments indicated that a specific transport system exists for L-glutamate and L-aspartate in the brushborder membrane, which does not transport neutral or basic amino acids. The uptake of both L-amino acids from the lumen into the cell was found to be rheogenic, probably reflecting the cotransport of two Na+ ions together with one amino acid molecule. The transport system has a slightly greater affinity for L-glutamate, but transports the smaller L-aspartate somewhat faster. Besides the L-isomers also D-glutamate and D-aspartate were found to depolarize the tubular cells which suggests that also the D-isomers are absorbed in the tubule, however they do not seem to use the same transport system as the L-isomers. In addition to the transport system in the brushborder, a similar Na+-dependent, rheogenic transport system for L-glutamate and L-aspartate was also found in the peritubular cell membrane, as deduced from cell cell depolarizations in response to these substrates applied peritubularly. The simultaneous presence of Na-driven transport systems in the apical and basal cell membrane which is not found with other amino acids, may explain the high intracellular accumulation of L-glutamate and L-aspartate in the kidney and provides a rational basis for explaining clinically observed cases of dicarboxylic aminoacidurias

Electrophysiological analysis of rat renal sugar and amino acid transport. II. Dependence on various transport parameters and inhibitors

Transepithelial and cellular electrical potential changes were measured in response to luminal perfusion of D-glucose and related substrates in micropuncture experiments on rat kidney in vivo. By studying the dependence of the potential response on various experimental parameters, some insight was obtained into the mechanism of Na+ coupled glucose absorption. The experiments confirm the driving forces for glucose absorption in the living cell to be: a) the Na concentration gradient, b) the electrical potential gradient and c) the glucose concentration gradient across the brush-border membrane. Furthermore they describe the substrate specificity of the cotransport mechanism and the mechanism of inhibition of D-glucose transport by various inhibitors, such as phlorizin, harmaline and ouabain. The latter experiments suggest that the active Na+ pump in the peritubular cell membrane, which establishes the Na+ ion gradient and the electrical potential gradient across the brushborder, contributes a measurable partial conductance to the overall electrical conductance of the peritubular cell membrane

pH-dependence of phosphate absorption in rat renal proximal tubule

Proximal tubular cell membrane potentials were measured in rat kidney in vivo and the response to luminal perfusion of 2mmolar phosphate (Pi) was studied. Pi transport was preferentially rheogenic at low pH (cotransport of 1H2PO4- plus 2Na+) but preferentially electroneutral at high pH (cotransport of 1HPO4-- plus 2Na+). The potential response as a function of pH conformed to a model which transports both H2PO4- and HPO4-- indiscriminately and whose maximal transport capacity increases with increasing pH. Further kinetic experiments are required to definitely exclude separate transport systems for both ionised forms. Hypercapnic phosphaturia may be explained by a decreased maximal transport capacity of Pi at low luminal pH

The stoichiometry of Na⁺ coupled anion absorption across the brushborder membrane of rat renal proximal tubule

Publisher Summary: This chapter investigates the electrical signals associated with Na-coupled anion absorption across the brush border membrane of rat kidney proximal tubular cells in vivo with the help of classical electrophysiological techniques. It has been found that single-charged anions, such as aspartate, glutamate, lactate, pyruvate, and primary phosphate, are transported with a surplus of positive charges, resulting most likely from the cotransport of two Na ions. The transport of some double-charged anions, such as secondary phosphate and sulfate is electroneutral, indicating a stoichiometry of Na+ to anion coupling of 2:1. Other double-charged ions, such as malonate, glutarate, α-keto-glutarate, and succinate generate positive cotransport currents and are transported together with three Na+ ions. The observations of electroneutral transport and particularly the observed change from rheogenic to electroneutral transport with the number of negative charges on the substrate suggest that the coupling ratios of the respective cotransport systems are integer numbers at least under experimental in vivo conditions